Risk factors for breast cancer, including a long menstrual history, obesity after menopause, use of hormone replacement therapy, nulliparity, late age at first pregnancy, and high serum or urinary estrogen levels, and experimental evidence, suggest that it is associated with prolonged or altered estrogen exposure. Estradiol (E2) is oxidatively biotransformed to catechol estrogens (CEs). CEs are oxidized to quinones which form mutagenic adducts with adenine and guanine in DNA and participate in redox cycling processes that lead to oxidative DNA damage. Breast tissue CE levels in breast tissue are 10 to 20 pmoles/g, and mounting evidence supports a role for CE in breast cancer. CEs are primarily inactivated by O-methylation catalyzed by catechol-O-methyltransferase (COMT). COMT is polymorphic and 25% of Caucasian Americans are homozygous for low activity COMT (COMTLL), which is also thermolabile. In a genetic epidemiology study, we found that COMTLL genotype confers a significantly increased risk for breast cancer in postmenopausal women with a high body mass index. This hypothesis driven epidemiology study was the fist to suggest that polymorphism in an enzyme involved in the inactivation of a reactive estrogen metabolite is associated with an increased risk for breast cancer. Subsequently, there have been 7 other epidemiology studies with mixed results demonstrating the need for increased understanding of the mechanisms of effects of CEs and of COMT in protection from their potential adverse effects. During the current project period we made the following observations: 1) COMT is highly protective against oxidative DNA damage caused by CEs in MCF-7 cells; 2) there are no intrinsic kinetic differences between the COMTL and COMTH activity enzymes; 3) the COMTL enzyme activity phenotype in human tissue cytosol is due to less enzyme protein; 4) thermolability studies on COMTL suggests that its instability may be due to its altered association with a cellular protein present; and 5) certain folate pathway micronutrients mediate the association between COMT genotype and breast cancer risk. The goal of this project remains "to conduct a rigorous experimental investigation of the hypothesis that decreased COMT activity results in increased DNA damage that contributes to increased cell transformation and breast cancer. The specific aims for the next project period are to: 1) determine the role of COMT in mouse mammary gland development and tumorigenesis using a COMT knockout mouse; 2) Confirm the relationship among COMT genotype, activity, and reduced COMT protein levels in extracts of human breast tissue; 3) define the mechanisms causing reduced levels of COMTL activity enzyme protein.